MX2012011647A

MX2012011647A - Selection and use of host cells for production of glycoproteins.

Info

Publication number: MX2012011647A
Application number: MX2012011647A
Authority: MX
Inventors: Brian E Collins; Jay Duffner; Victor Farutin; Naveen Bhatnagar; Lakshmanan Thiruneelakantapillai; Carlos J Bosques; Ganesh Venkataraman
Original assignee: Momenta Pharmaceuticals Inc
Priority date: 2010-04-07
Filing date: 2011-04-07
Publication date: 2012-11-29
Also published as: WO2011127325A1; AU2011237445A1; CN102917733A; US20130123126A1; EP2555801A4; AU2011237445B2; CA2794709A1; BR112012025654A2; EP2555801A1

Abstract

A method of making a glycoprotein having a selected glycostructure.

Description

SELECTION AND USE OF GUEST CELLS FOR PRODUCTION OF GLYCOOPROTEINS FIELD OF THE INVENTION The invention is directed to methods of selecting host cells for the production of glycoproteins, host cells and other methods, cells and related glycoproteins.

BACKGROUND OF THE INVENTION A typical glycoprotein product differs significantly in terms of the complexity of a typical small molecule drug. The sugar structures attached to the amino acid skeleton of a glycoprotein can vary structurally in many ways, including sequence, branching, sugar content and heterogeneity. In this way, the glycoprotein products may be complex heterogeneous mixtures of several structurally diverse molecules that themselves have complex glycan structures. Glycosylation not only increases the structural complexity of the molecule, but also affects or conditions several of the biological and clinical attributes of a glycoprotein.

SUMMARY OF THE INVENTION The appearance of post-translational modifications, for example, glycostructures, glycan complement, glycan component, in proteins, is the Ref.:236024 result of an extremely complex interaction of many factors. The methods described herein depend, in part, on multi-observational analysis of the character of the post-translational modifications, for example, glycostructures, glycan complement, glycan component, in the proteins made from the selected cell populations. The methods allow comparisons of the capacity of different cell populations in terms of their ability to confer complicated post-translational modifications, for example, glycostructures, glycan complement, glycan component, in the proteins they perform. The profiles of the quality attribute of the cell population provide surprisingly important distinctions between cell populations, even for very similar cell lines. Accordingly, the methods described herein may be used to select an appropriate host cell for the production of an objective glycoprotein (eg, the production of a biosimilar or biogeneric product of a commercially available biological therapeutic glycoprotein), eg, the methods described they may be used herein to identify and / or select a host cell for the production of a biosimilar or biogeneric product that best matches the glycosylation properties of the host cell in which the commercially available biological glycoprotein was produced, for example, in cases where the population of host cells in which the commercial therapeutic biological glycoprotein is produced is unknown by the manufacturer of the biosimilar or biogeneric product. In aspects, a host cell population suitable for the production of an objective glycoprotein is selected using the methods described herein.

In one aspect, the invention features a method of modeling a glycoprotein having a selected post-translational modification (eg, a glycostructure, glycan complement, selected glycan component, eg, with a selected glycan structure), or which provides or selects a cell population, eg, a population of CHO cells, for example, to use in the embodiment a glycoprotein having a selected post-translational modification (eg, a glycostructure, glycan complement, selected glycan component) , for example, with a selected glycan structure). The method includes: (a) acquiring, directly or indirectly, the identity of a cell population for the production of the glycoprotein, wherein the identity is acquired or determined by a method described herein, for example, by (i) acquisition, for each of a plurality of isolates or aliquots of a first cell population, of a value that is expressed in terms of a post-translational modification, that value being a function of a plurality of different observations (e.g. , the level of expression of a plurality of different genes or the level of expression of a plurality of different glycostructures, glycan structures, glycan components or combinations thereof) to provide a set of values for the first cell population; (ii) acquisition, for each of a plurality of isolates or aliquots of a second cell population, of a value that is expressed in terms of a post-translational modification, that value being a function of a plurality of different observations (e.g. , the level of expression of a plurality of different genes or the level of expression of a plurality of different glycostructures, glycan structures, glycan components or combinations thereof) to provide a set of values for the second cell population; (iii) comparison of a value for a selected post-translational modification (for example, a glycostructure, glycan complement, glycan component or combinations of these) with the set of values for the first cell population and with the set of values for the second cell population; Y (iv) in response to the comparison, selection of the first or second cell population.

In one embodiment, the method is a method for providing or selecting a cell population, for example, a population of CHO cells, for example, for use in the form of a glycoprotein having a second post-translational modification (e.g., a glycostructure, glycan complement, selected glycan component, for example, with a selected glycan structure) and the method further comprises (b) culturing the selected cell population.

In one embodiment, the method is a method of modeling a glycoprotein having a selected post-translational modification (eg, a glycostructure, glycan complement, selected glycan component, eg, with a selected glycan structure) and the The method further comprises (b) performing a glycoprotein having a selected post-translational modification (eg, glycostructure, glycan complement or glycan component, eg, with a selected glycan structure) in the selected cell population.

In one embodiment, the method may further comprise genetically modifying the identified cell population to express the glycoprotein, for example, by introducing a nucleic acid encoding all or part of the glycoprotein in the cell population identified prior to step (b).

In one embodiment, a set of values is acquired for a plurality, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, of cell populations.

In one embodiment, each of the cell populations in the plurality is of the same species, tissue and cell type, although in modalities they may differ by naturally acquired or intentionally induced mutations.

In some embodiments, each of the cell populations in the plurality derives from a different cell line.

In one embodiment, each of the cell populations in the plurality derives from a single cell clone different from a specific cell line.

In one embodiment, each of the cell populations in the plurality is an intimately related cell population.

In one embodiment, each cell population of the cell populations shares a common predecessor cell wherein the predecessor cell was not part of an organism, for example, the predecessor cell was a cultured cell or a founding cell of a cell line. Typically, the common predecessor cell is a cell, eg, a cultured cell, that has been removed from a multicellular organism, eg, an insect or animal, eg, a mammal or primate, excluding as a common predecessor cell, precursor cells. of the animal or predecessors of the animal from which the common predecessor cell is taken.

In one embodiment, each of the cell populations is derived from a common predecessor cell and none of the cell populations of the plurality has an intentionally induced mutation that inactivates a gene that encodes a protein that synthesizes, binds or modifies a glycan. In one embodiment, each of the cell populations of the plurality derives from a common predecessor cell and none of the cell populations of the plurality has an inactivation mutation intentionally induced in a gene encoding a protein selected from: a glycosyltransferase (e.g. , MGAT1 (GlcNAc TI), alpha mannosidase II, IIx, alpha mannosidase IB, alpha mannosidase IA, FucTl-9, glucosidase (eg, GCS1, GAAB), a precursor of biosynthesis or localization or trafficking, GNE (eg, glucosamine (UDP-N-acetyl) -2-epimerase / N-acetyl mannosamine), UDP phosphatase of the Golgi apparatus, UDP-GlcNAc transporter, UAP-1 (UDP-N-acetylhexosamine pyrophosphorylase), PGM-3-phosphoglucomutase 3, NAGK -N-acetyl-D-glucosamine kinase, GNPNAT1 - glucosamine-phosphate N-acetyltransferase 1, UGP-2 -UDP-glucose pyrophosphorylase 2, UGDH - UDP-glucose 6-dehydrogenase, GA1K-1 - Galactocinase-1, PGM-1 Fosfoglucomutase-1, GCK - glucokinase), a target for altering r the location or traffic through the ER and the Golgi apparatus, eg, a chaperone (BiP, SNARE, cpn, hsp), EDEM (mannase-type degradation protein of the ER), MANEA, mannose receptor. In one embodiment, each of the cell populations of the plurality derives from a common predecessor cell and none of the cell populations of the plurality has an intentionally induced inactivation mutation that modulates the level of a glycan metabolite, e.g., a metabolite described in the present.

In one embodiment, the cell populations do not derive from a Pro-5 cell line. In one embodiment, the cell populations are not modified (for example, they are not chemically mutagenized) to be resistant to a lectin.

In one embodiment, the selected post-translational modification is a glycan supplement or selected glycan component.

In one embodiment, the glycoprotein is a biological therapeutic product, for example, a therapeutic antibody, Fe receptor fusion protein, hormone, cytokine. In one embodiment, the glycoprotein is a biosimilar or biogeneric version of a marketed therapeutic biological product.

In one embodiment, observations for each cell population include at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more observations of gene expression levels. In one embodiment, the observations for each cell population include at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more observations relative to the levels of a glycan metabolite.

In one embodiment, the method, for example, (i) - (iv) comprises: (i) acquire a profile of the cell population quality attribute (a profile) comprising a set of responses, where a response is expressed in terms of a post-translational modification and is the product of an operation by a plurality of observations, for each of a plurality of cell populations, the acquired profiles forming a plurality of different profiles; (ii) acquire the identity of a selected post-translational modification (eg, glycostructure, glycan complement, glycan component or a combination of these), - (ii) compare the acquired profile with the identity acquired in (ii); (iv) in response to the comparison (for example, when the acquired profile and the identity acquired in (ii) show a preselected relationship with each other, for example, the former includes the latter), select one of the plurality of cell populations for production of the object glycoprotein; select or provide the cell population to perform the glycoprotein and / or perform the glycoprotein having the selected post-translational modification (eg, glycostructure) in the selected cell population. In some embodiments, the method further comprises introducing a nucleic acid encoding all or part of the glycoprotein in the identified cell population.

In one embodiment, the identity of the cell population is acquired directly.

In one embodiment, the identity of the cell population is acquired indirectly.

In one modality, the dimensionality of a response is smaller than the dimensionality of the observations.

In one embodiment, the method comprises a manipulation that reduces the dimensionality of the response, compared to the number of observations.

In one modality, the comparison is made with the answer ', where the answer1 has at least one dimension smaller than the answer.

In one embodiment, the method comprises a manipulation that reduces the dimensionality of a response, as compared to a response.

In some embodiments, an underlying observation is expressed in terms of glycan structure, glycostructure, glycan component, or glycan complement. The modality can have one or more of the following properties: the responses in the acquired profile are based on a first and a second observation and the first observation is the level of a first post-translational modification, for example, glycan structure, glycostructure, glycan component or glycan complement and the second observation is the level of a second post-translational modification, eg, glycan structure, glycostructure, glycan component or glycan complement; the comparison comprises comparing the selected post-translational modification, eg, glycostructure, with a dimensional representation of the plurality of profiles wherein the axis in each dimension represents a different aspect of glycostructure, glycan complement or glycan component, for example, wherein the axis for a first dimension represents the level of glycan A and the axis for a second dimension represents the level of glycan B.

In some embodiments, an underlying observation is not expressed in terms of glycan structure and is expressed, for example, in terms of the level of expression of one or more genes. In the modalities, the operation not only gives an answer but also offers an answer in terms of the structure of glycan. The modality can have one or more of the following properties: the responses in the acquired profile are based on a first and second observation and at least one of the first and second observation is not expressed in terms of post-translational structure, for example, glycostructure, but is expressed in terms of a parameter related to the post-translational structure, eg, glycostructure, and the operation provides a response expressed in terms of post-translational structure, eg, glycostructure, glycan complement or glycan component; the responses in the acquired profile are based on a first and second observation and the first observation is the level of a first metabolite and the second observation is the level of a second metabolite; the comparison comprises comparing the response for the glycostructure, glycan complement or selected glycan component with a n-dimensional representation of the plurality of different acquired profiles where the axis in each dimension is correlated with a different aspect of glycan, glycan complement or component of glycan, for example, where the axis for a first dimension correlates with the level of glycan A and the axis for a second dimension correlates with the level of glycan B.

The method requires stages of "acquisition", for example, acquisition of a profile or acquisition of the identity of a selected post-translational modification. The acquisition of the method can include one of several elements.

In one embodiment, acquiring a value involves subjecting a sample to a process that results in a physical change in the sample or other substance, for example, an analytical reagent or a device used in the analysis. The methods comprise analytical methods, for example, a method that includes one or more of the following: separating a substance, for example, an analyte or a fragment or other derivative thereof, from another substance; combining an analyte or fragment or other derivative thereof, with another substance, for example, a buffer, solvent or reagent; or changing the structure of an analyte or a fragment of another derivative thereof, for example, breaking or forming a covalent or non-covalent bond, between a first and a second atom of the analyte or reagent.

In other embodiments, for example, in embodiments where the method includes the production of a glycoprotein, or the cultivation of a cell, the harvesting of a glycoprotein or the purification of a glycoprotein, or another step resulting in a transformation of a used entity in the method, for example, a cell, glycoprotein or reagent, the acquisition step may be a subsequent step that can be provided without transformation, for example, by inspection, comparison or receipt of information from another party.

In one modality, acquiring a profile involves performing a chemical or physical analysis to determine the profile.

In one modality, acquiring a profile involves receiving information regarding the profile of another party.

In one modality, acquiring the identity of a post-translational modification involves performing a chemical or physical analysis to determine the identity.

In one embodiment, acquiring the identity of a post-translational modification comprises selecting the identity of a description of a drug, for example, of a prospect.

In one modality, acquiring the identity of a post-translational modification comprises selecting the identity of a list or table.

In one modality, acquiring the identity of a post-translational modification comprises receiving information regarding the identity of the post-translational modification of another party.

As described elsewhere herein, an observation can be expressed in terms of glycan structure.

In one modality, one observation is the level of 4.4.1.0.0.

In one modality, one observation is the level of 4,4,1,1,0.

In one modality, one observation is the level of 4,5,1,0,0.

In one modality, an observation is the level of 4,5,1,1,0.

In one modality, one observation is the level of 4,5,1,2,0.

In one modality, one observation is the level of 5.5.1.0.0.

In one modality, one observation is the level of 5,6,1,0,0.

In one modality, one observation is the level of 5,6,1,1,0.

In one modality, an observation is the level of 5,6,1,2,0.

In one modality, one observation is the level of 5,6,1,3,0.

In one modality, one observation is the level of 6, 6.1, 1.0.

In one modality, one observation is the level of 6,6,1,2,0.

In one modality, an observation is the level of 6,7,1,1,0.

In one modality, an observation is the level of 6,7,1,2,0.

In one modality, an observation is the level of 6,7,1,3,0.

In one modality, one observation is the level of 6,7,1,4,0.

As described elsewhere herein, an observation may be expressed in terms other than the structure of glycan, glycan complement or glycan component. In one modality, one observation is the level of gene expression.

In one embodiment, one observation is the level of expression of a glycosyltransferase.

In one embodiment, an observation is the level of expression of a gene involved in glycan biosynthesis.

In one modality, an observation is the level of a metabolite.

In one modality, one observation is the UMP level. In one modality, one observation is the GTP level. In one modality, one observation is the UDP-Gal level.

In one mode, one observation is the GDP-Fuc level.

As described elsewhere herein, the methods described herein can be used with a range of cell populations, for example, cell strains different from a parental cell line or isolated from a parental cell strain.

In one embodiment, one of the cell populations of the plurality of cell populations is a line of CHO cells.

In one embodiment, one of the cell populations of the plurality of cell populations is a cell line CHO Kl.

In one embodiment, one of the cell populations of the plurality of cell populations is a CHO S cell line.

In one embodiment, one of the cell populations of the plurality of cell populations is a cell line DG44.

In one embodiment, one of the cell populations of the plurality of cell populations is a DHFR (-) cell line.

In one embodiment, one of the cell populations of the plurality of cell populations is a CHO GS cell line.

As described elsewhere in the methods described herein, various types of operation are suitable for use in the methods.

In one embodiment, the operation is an arithmetic combination of a plurality of observations.

In one embodiment, the operation is an adjustment to a model of a plurality of observations.

In one modality, the model is a linear model.

In one embodiment, the operation comprises relating, for example, associating, correlating or matching values for observations derived from a source of information, for example, a list, table or database, for example, a publicly available database. .

As described elsewhere in the methods described herein, various types of responses are suitable for use in the methods.

In one embodiment, the response is the product of an operation at the level of expression of a plurality of genes, for example, wherein: at least one of the plurality of genes encodes a protein that forms the selected post-translational modification; at least one of the plurality of genes encodes a protein that reduces the level of selected post-translational modification; the answer is the product of an operation at the ST3GAL3 and ST3GAL levels.

In another aspect, the invention features, a method for providing or selecting a cell population of a plurality of isolates of the same cell type, for example, isolated from a population of CHO cells, for example, for use in the form of a glycoprotein which has a post-translational modification (eg, a glycan, glycan complement or glycan component selected, for example, with a selected glycan structure). The method includes: (a) acquire the identity of a selected post-translational modification (eg, glycostructure, glycan complement, glycan component, for example, with a selected glycan structure), (b) acquiring an evaluation, for example, by using the method described herein, the ability of each of the plurality of isolates of the cell type to produce the selected post-translational modification, (c) selecting an isolate from the plurality of isolates, (d) optionally culturing the selected cell population; thus providing a cell population.

In one embodiment, the method also comprises (b) culturing the selected cell population.

In one embodiment, the method further comprises (b) performing a glycoprotein having a selected post-translational modification (eg, glycostructure, glycan complement or glycan component, eg, with a selected glycan structure) in the cell population selected In one embodiment, the method may further comprise genetically modifying the identified cell population to express the glycoprotein, for example, by introducing a nucleic acid encoding all or part of the glycoprotein in the cell population identified prior to step (b).

In some embodiments, each of the cell populations in the plurality derives from a different cell line, different cell strain or different clone.

In one embodiment, each of the cell populations is derived from a common predecessor cell and none of the cell populations of the plurality has an intentionally induced mutation that inactivates a gene that encodes a protein that synthesizes, binds or modifies a glycan. In one embodiment, each of the cell populations of the plurality derives from a common predecessor cell and none of the cell populations of the plurality has an inactivation mutation intentionally induced in a gene encoding a protein selected from: a glycosyltransferase (e.g. , MGAT1 (GlcNAc TI), alpha mannosidase II, IIx, alpha mannosidase IB, alpha mannosidase IA, FucTl-9, glucosidase (for example, GCS1, GANAB), a precursor of biosynthesis or localization or trafficking, GNE (for example, glucosamine (UDP-N-acetyl) -2-epimerase / N-acetylmannosamine), UDP phosphatase of the Golgi apparatus, UDP-GlcNAc transporter, UAP-1 (UDP-N-acetylhexosamine pyrophosphorylase), PGM-3-phosphoglucomutase 3, NAGK -N-acetyl-D-glucosamine kinase, GNPNATl - glucosamine-phosphate N-acetyltransferase 1, UGP-2 -UDP-glucose pyrophosphorylase 2, UGDH - UDP-glucose 6-dehydrogenase, GA1K-1 - Galactocinasa-1, PGM-1 Fosfoglucomutase-1, GCK - glucokinase), an objective to alter localization or trafficking through the ER and the Golgi apparatus, for example, a chaperone (BiP, SNARE, cpn, hsp), EDEM (mannase-like protein of degradation of the ER), MANEA, mannose receptor. In one embodiment, each of the cell populations of the plurality derives from a common predecessor cell and none of the cell populations of the plurality has an intentionally induced inactivation mutation that modulates the level of a glycan metabolite, e.g., a metabolite described in the present.

In one embodiment, the observations for each of the cell populations include at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more observations of gene expression levels. In one embodiment, the observations for each of the cell populations include at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more observations relative to the levels of a glycan metabolite.

In one embodiment, the method, for example, (i) - (iv) comprises: (i) acquire a profile of the cell population quality attribute (a profile) comprising a set of responses, where a response is expressed in terms of a post-translational modification and is the product of an operation by a plurality of observations, for each of a plurality of cell populations, the acquired profiles forming a plurality of different profiles; (ii) acquiring the identity of a selected post-translational modification (eg, glycostructure, glycan complement or glycan component); (iii) compare the acquired profile with the identity acquired in (ii); (iv) in response to the comparison (for example, when the acquired profile and the identity acquired in (ii) show a preselected relationship with each other, for example, the former includes the latter), select one of the plurality of cell populations for production of the white glycoprotein; select or provide the cell population to perform the glycoprotein and / or perform the glycoprotein having the selected post-translational modification (eg, glycostructure) in the selected cell population. In some embodiments, the method further comprises genetically modifying the identified cell population to express the glycoprotein, for example, by introducing a nucleic acid encoding all or part of the glycoprotein in the identified cell population.

In one embodiment, the identity of the cell population is acquired directly.

In one embodiment, the identity of the cell population is acquired indirectly.

In one modality, the dimensionality of a response is less than the dimensionality of the observations.

In one modality, the comparison is made with the answer ', where the answer' has at least one dimension smaller than the answer.

In some embodiments, an underlying observation is not expressed in terms of glycan structure and is expressed, for example, in terms of the level of expression of one or more genes. In the modalities, the operation not only gives an answer but also offers an answer in terms of the structure of glycan. The modality can have one or more of the following properties: the responses in the acquired profile are based on a first and second observation and at least one of the first and second observations are not expressed in terms of post-translational structure, for example, glycostructure, but are expressed in terms of a parameter related to the post-translational structure, eg, glycostructure, and the operation provides a response expressed in terms of post-translational structure, eg, glycostructure, glycan complement or glycan component; the responses in the acquired profile are based on a first and second observation and the first observation is the level of a first metabolite and the second observation is the level of a second metabolite; the comparison comprises comparing the response for the selected glycostructure, glycan complement or glycan component with a n-dimensional representation of the plurality of different acquired profiles where the axis in each dimension correlates with a different aspect of glycostructure, glycan complement or component of glycan, for example, wherein the axis for a first dimension correlates with the level of glycan A and the axis for a second dimension correlates with the level of glycan B.

The method requires the steps of "acquisition", for example, acquisition of a profile or acquisition of the identity of a selected post-translational modification. The acquisition of the method can include one of several elements.

In one modality, one observation is the level of 4.4.1.0.0.

In one modality, one observation is the level of 4,4,1,1,0.

In one modality, one observation is the level of 4,5,1,0,0.

In one modality, one observation is the level of 4,5,1,1,0.

In one modality, an observation is the level of 4,5,1,2,0.

In one modality, one observation is the level of 5.5.1.0.0.

In one modality, one observation is the level of 5,6,1,0,0.

In one modality, one observation is the level of 5,6,1,1,0.

In one modality, one observation is the level of 5,6,1,2,0.

In one modality, an observation is the level of 5,6,1,3,0.

In one modality, one observation is the level of 6,6,1,1,0.

In one modality, one observation is the level of 6,6,1,2,0.

In one modality, one observation is the level of 6,7,1,1,0.

In one modality, one observation is the level of 6,7,1,2,0.

In one modality, an observation is the level of 6,7,1,3,0.

In one modality, one observation is the level of 6,7,1,4,0.

In one modality, an observation is the level of a metabolite.

In one modality, one observation is the UMP level. In one modality, one observation is the GTP level. In one embodiment, one observation is the UDP-Gal level.

In one mode, one observation is the GDP-Fuc level.

As described elsewhere herein, the methods described herein can be used with a range of cell populations, for example, cell strains different from a parental cell line or different isolates from a parental cell strain.

In one embodiment, one of the cell populations of the plurality of cell populations is a line of CHO Kl cells.

In one embodiment, one of the cell populations of the plurality of cell populations is a line of CHO S cells.

In one modality, the model is a linear model. In one embodiment, the operation comprises relating, for example, associating, correlating or matching values for observations derived from a source of information, for example, a list, table or database, for example, a publicly available database. .

In one embodiment, the response is the product of an operation at the expression level of a plurality of genes, for example, wherein: at least one of the plurality of genes encodes a protein that forms the selected post-translational modification; at least one of the plurality of genes encodes a protein that reduces the level of selected post-translational modification; the answer is the product of an operation at the ST3GAL3 and ST3GAL4 levels.

In another aspect, the invention features a method of selecting or evaluating a cell, for example, for use in the mode of a glycoprotein having a selected post-translational modification. The method includes: (a) acquire, directly or indirectly, the identity of a cell population for the production of the glycoprotein, where the acquired identity is determined by (i) acquisition, for each of a plurality of isolates or aliquots of a first cell population, of a value that is expressed in terms of a post-translational modification, that value being a function of a plurality of different observations (e.g. , the level of expression of a plurality of different genes or the level of expression of a plurality of different glycostructure, glycan complement or glycan component) to provide a set of values for the first cell population; (ii) acquisition, for each of a plurality of isolates or aliquots of a second cell population, of a value that is expressed in terms of a post-translational modification, that value being a function of a plurality of different observations to provide a set of values for the second cell population; (iii) comparison of a value for a post-translational modification (eg, a glycostructure, a glycan complement or a glycan component, for example, with a selected glycan structure) with the set of values for the first cell population and with the set of values for the second cell population; Y (iv) in response to the comparison, selection of the first or second cell population; or select in that way or evaluate the cell, where: (1) stage (i and / or ii) comprises cultivating a cell population, performing a chemical or physical analysis to provide a response, for example, chemical or physical analysis to provide an observation.

In one embodiment, the method, for example, (i) - (iv) comprises: (i) acquire a profile of the quality attribute of the cell population that comprises a set of responses, where a response is expressed in terms of a post-translational modification and is the product of an operation by a plurality of observations, for each one of a plurality of cell populations, the acquired profiles forming a plurality of distinct profiles; (ii) acquiring the identity of a selected post-translational modification (eg, glycostructure, glycan complement or glycan component); (iii) compare the acquired profile with the identity acquired in (b); (iv) in response to the comparison (for example, when the acquired profile and the identity acquired in (b) show a preselected relationship with each other, for example, the former includes the latter), select one of the plurality of cell populations for production of the object glycoprotein, to select in that way or evaluate the cell, where: step (i) comprises cultivating a cell population, performing a chemical or physical analysis to provide a response, for example, a chemical or physical analysis to provide an observation; step (ii) comprises performing a chemical or physical analysis to provide the identity; step (iii) comprises providing a profile representation as a n-dimensional space and comparing the identity with the space; or optionally, the method further comprises culturing the selected cell.

As described elsewhere herein, an observation may be expressed in terms of glycostructure, glycan complement or glycan component, for example, with a selected glycan structure, for example, a glycan structure described herein.

As described elsewhere herein, an observation can be expressed in terms other than glycostructure, glycan complement or glycan component, for example, with a glycan structure selected, eg, the level of gene expression, for example. , a gene described herein or a metabolite, for example, a metabolite described herein.

As described elsewhere in the methods described herein can be used with a range of cell populations, for example, a population of CHO cells or another cell population described herein.

As described elsewhere in the methods described herein, various types of operation are suitable for use in the methods, for example, operations described herein, for example, an arithmetic combination or linear model.

As described elsewhere in the methods described herein several types of responses are suitable for use in the methods, for example, a response described herein, for example, a response that is the product of an operation at the level of expression of a plurality of genes.

As described elsewhere herein, the types of responses and / or observations may be the level of expression of a gene or genes described herein.

In another aspect, the invention features a method for providing a population of cells, for example, for use in the mode of a glycoprotein having a selected post-translational modification. The method includes: (a) acquire a profile of the quality attribute of the cell population comprising a set of responses, where a response is expressed in terms of a post-translational modification and is the product of an operation by a plurality of observations, for each one of a plurality of cell populations, the acquired profiles forming a plurality of distinct profiles; (ii) acquiring the identity of a selected post-translational modification (eg, glycostructure, glycan complement or glycan component, eg, with a selected glycan structure); (c) compare the acquired profile with the identity acquired in (b); (d) in response to the comparison (for example, when the acquired profile and the identity acquired in (b) show a preselected relationship with each other, for example, the former includes the latter), select one of the plurality of cell populations for production of the object glycoprotein; and (e) cultivating the selected cell population to provide the population.

As described elsewhere herein, a method may require one or more "acquisition" steps, eg, acquisition of a profile or acquisition of the identity of a selected post-translational modification. In one embodiment acquiring a value comprises subjecting a sample to a process that results in a physical change in the sample or other substance, for example, an analytical reagent or a device used in the analysis, for example, an analysis described herein. In other embodiments, for example, in embodiments where the method includes the production of a glycoprotein, or the cultivation of a cell, the harvesting of a glycoprotein or the purification of a glycoprotein or another step resulting in a transformation of an entity used in the method, for example, a cell, glycoprotein or reagent, the acquisition step being a subsequent step that can be provided without transformation, for example, by inspection, comparison or receipt of information from another party.

As described elsewhere herein, an observation may be expressed in terms that are not glycan structure, eg, the level of gene expression, eg, a gene described herein or a metabolite, eg, a metabolite. described in the present.

As described elsewhere in the methods described herein, various types of responses are suitable for use in the methods, for example, a response described herein, for example, a response that is the product of an operation in the level of expression of a plurality of genes.

In another aspect, the invention features a method for monitoring a production process to perform a glycoprotein having a selected post-translational modification. The method includes: (a) acquiring, for each of a plurality of isolates or aliquots of a first cell population, a value that is expressed in terms of a post-translational modification, that value being a function of a plurality of different observations (e.g., the level of expression of a plurality of different genes or the level of expression of a plurality of different glycostructure, glycan complement or glycan component, for example, with a selected glycan structure) to provide a set of values for the first population cell phone; (b) comparing a value for a selected post-translational modification (eg, a glycostructure, a glycan complement or a glycan component, eg, with a selected glycan structure) with the set of values for the first cell population; Y (c) if the comparison shows a first preselected relationship with the set of values, for example, the set of values includes the identity, look for a first option, for example, continue with the cultivation; and if the comparison shows a second preselected relationship with the set of values, for example, the set of values does not include the identity, look for a second option, for example, cease the current cultivation conditions or cultivate under a new set of conditions.

In one embodiment, the method comprises: (a) acquire a profile of the cellular population quality attribute that comprises a set of responses, where a response is expressed in terms of a post-translational modification and is the product of an operation by a plurality of observations, for an aliquot of production cells; (b) comparing the identity of a selected post-translational modification (eg, glycostructure) with the profile; (c) if the comparison shows a first preselected relation with the profile, for example, the profile includes the identity, look for a first option, for example, continue with the cultivation; and if the comparison shows a second preselected relationship with the profile, for example, the profile does not include the identity, look for a second option, for example, cease the current cultivation conditions or cultivate under a new set of conditions.

In one embodiment, the glycan component and / or glycan complement selected is a glycan component and / or glycan complement of a biological therapeutic glycoprotein, eg, a commercially available biological therapeutic glycoprotein, and if the profile includes the identity of the component. of glycan and / or glycan complement selected to continue culturing the CHO cells, for example, to produce a biogeneric or biosimilar glycoprotein of the biological therapeutic glycoprotein.

In one embodiment, the glycan component and / or glycan complement selected is a glycan component and / or glycan complement of a biological therapeutic glycoprotein, eg, a commercially available biological therapeutic glycoprotein, and if the profile does not include the identity of the glycan component and / or glycan complement selected look for a second option, for example, selecting a different CHO cell population that has a profile that includes the glycan component and / or glycan complement selected, for example, to produce a glycoprotein biogeneric or biosimilar of the biological therapeutic glycoprotein.

In one embodiment, the glycan component and / or glycan complement selected is a glycan component and / or glycan complement of a biological therapeutic glycoprotein, eg, a commercially available biological therapeutic glycoprotein, and if the profile includes the identity of the component. of glycan and / or glycan complement selected to continue culturing the CHO cells, for example, to produce the biological therapeutic glycoprotein.

In one embodiment, the glycan component and / or glycan complement selected is a glycan component and / or glycan complement of a biological therapeutic glycoprotein, eg, a commercially available biological therapeutic glycoprotein, and if the profile does not include the identity of the Glycan component and / or glycan supplement selected look for a second option, for example, cease current culture conditions or grow under a new set of conditions, for example, conditions that result in a profile that includes the identity of the glycan component and / or selected glycan complement, to produce the biological therapeutic glycoprotein.

As described elsewhere herein, a method may require one or more "acquisition" steps, eg, acquisition of a profile or acquisition of the identity of a selected post-translational modification. In one embodiment, acquiring a value comprises subjecting a sample to a process that results in a physical change in the sample or other substance, for example, an analytical reagent or a device used in the analysis, for example, an analysis described herein. . In other embodiments, for example, in embodiments where the method includes the production of a glycoprotein, or the cultivation of a cell, the harvesting of a glycoprotein or the purification of a glycoprotein or another step resulting in a transformation of an entity used in the method, for example, a cell, glycoprotein or reagent, the acquisition step being a subsequent step that can be provided without transformation, for example, by inspection, comparison or receipt of information from another party.

As described elsewhere herein, an observation can be expressed in terms of glycan structure, for example, a glycan structure described herein.

As described elsewhere herein, the methods described herein may be used with a range of different populations, for example, a population of CHO or other cells described herein.

As described elsewhere in the methods described herein, various types of operation are suitable for use in the methods, for example, the operations described herein, for example, an arithmetic combination or linear model.

In another aspect, the invention provides a method of selecting a glycoprotein for manufacture in a cell population. The method includes: (a) acquiring a profile of the quality attribute of the cell population comprising a set of responses, wherein a response is expressed in terms of a glycostructure and is the product of an operation by a plurality of observations, for a cell population; (b) acquire the identities of a plurality of glycostructures; (c) compare the acquired profile with the identities acquired in (b); (d) in response to the comparison (for example, when the identities acquired in (b) and the acquired profile show a preselected relationship with each other, for example, the former includes the latter), selecting one of the plurality of glycostructures for production in the cell population; Y (e) carrying out a glycoprotein having the selected glycostructure in the cell population.

As described elsewhere herein, a method may require one or more "acquisition" steps, eg, acquisition of a profile or acquisition of the identity of a selected post-translational modification. In one embodiment acquiring a value comprises subjecting a sample to a process that results in a physical change in the sample or other substance, for example, an analytical reagent or a device used in the analysis, for example, an analysis described herein. In other modalities, for example, in embodiments where the method includes the production of a glycoprotein, or the cultivation of a cell, the harvesting of a glycoprotein or the purification of a glycoprotein, or another step resulting in a transformation of an entity used in the method, for example , a cell, glycoprotein or reagent, the acquisition step may be a subsequent step that can be provided without transformation, for example, by inspection, comparison or receipt of information from another party.

In one aspect, the description presents a database, comprising a plurality of records for isolates from a cell population of a preselected cell population, eg, CHO cells, wherein each record comprises an identifier for a single isolate (as opposed to to others in the plurality) of the preselected cell type and an identifier for a profile of the quality attribute of the cell population for the isolate, and wherein the profile of the quality attribute of the cell population for each entry is unique (in opposition to others in plurality) for the isolated.

In one embodiment, a preselected cell type is CHO or another cell population described herein.

A database comprising a plurality of registers, each register of the plurality corresponding to an isolate of a cell population of a preselected cell population, for example, CHO cells, wherein the plurality of registers comprises: a first record comprising an identifier for a first isolate of the preselected cell type and an identifier for a first profile of the quality attribute of the cell population for the first isolate, a second record comprising an identifier for a second isolate of the preselected cell type and an identifier for a second profile of the quality attribute of the single cell population for the second isolate, wherein the profile of the quality attribute of the cellular population in each of the registers of the plurality of registers is different for each isolate in the plurality is different from the profile of the attribute of quality of the cellular population for each of the other isolates in the plurality.

In one embodiment, the database comprises records for at least 5, 10 or 20 isolates.

In one aspect, the invention features a method of modeling a glycoprotein having a selected glycan component and / or glycan complement, or providing or selecting a population of CHO cells from a plurality of CHO populations, for example, use in the form of a glycoprotein having a selected glycan component and / or glycan complement. The method includes: (a) acquiring, directly or indirectly, the identity of a population of CHO cells for the production of the glycoprotein, wherein the identity is acquired or determined by a method described herein, for example, by (i) acquisition, for each of a plurality of isolates or aliquots of a first population of CHO cells, for example, a population of CHO cells described herein, of a value that is expressed in terms of glycan component and / or glycan complement, the value being a function of a plurality of different observations that include the level of expression of a plurality of genes and the level of expression of a plurality of different glycostructures, glycan structures, glycan components, glycan complement or combinations thereof, to provide a set of values for the first population of CHO cells; (ii) acquisition, for each of a plurality of isolates or aliquots of a second population of CHO cells, for example, a population of CHO cells described herein, of a value that is expressed in terms of glycan component and / or glycan complement, the value being a function of a plurality of different observations that include the level of expression of a plurality of genes and / or metabolites and the level of expression of a plurality of different glycostructures, glycan structures, glycan components , complement of glycan or combinations of these, to provide a set of values for the second population of CHO cells, wherein the second population of CHO cells differs from the first population of CHO cells, for example, by a mutation naturally acquired or intentionally induced; (iii) comparing a value for a glycan component or selected glycan complement with the set of values for the first population of CHO cells and with the set of values for the second population of CHO cells; (iv) in response to the comparison, selection of the first or second population of CHO cells.

In one embodiment, the glycan component and / or glycan complement selected is a glycan component or glycan complement of a biological therapeutic glycoprotein, eg, a commercially available biological therapeutic glycoprotein, and the selected CHO cell population has a set of values indicating that it produces a glycoprotein having the glycan component and / or glycan complement of the marketed biological therapeutic glycoprotein.

In one embodiment, the glycoprotein is a therapeutic antibody, Fe receptor fusion protein, hormone, cytokine. In one embodiment, the glycoprotein is a biosimilar or biogeneric version of a marketed therapeutic biological product.

In one embodiment, the method is a method for providing or selecting a population of CHO cells, for example, for use in the mode of a glycoprotein having a selected post-translational modification (eg, a glycostructure, glycan complement, component of glycan selected, for example, with a selected glycan structure) and the method further comprises (b) culturing the selected CHO cell population.

In one embodiment, the method is a method of modeling a glycoprotein having a glycan complement and / or selected glycan component, and the method further comprises (b) performing a glycoprotein having a glycan complement and / or glycan component. selected glycan in the selected CHO cell population.

In one embodiment, the method may further comprise genetically modifying the population of CHO cells identified to express the glycoprotein, for example, by introducing a nucleic acid encoding all or part of the glycoprotein in the population of CHO cells identified prior to step (b) ).

In one embodiment, one of the CHO cell populations of the plurality of CHO cell populations is a CHO Kl cell line.

In one embodiment, one of the CHO cell populations of the plurality of CHO cell populations is a CHO S cell line.

In one embodiment, one of the CHO cell populations of the plurality of CHO cell populations is a DG44 cell line.

In one embodiment, one of the CHO cell populations of the plurality of CHO cell populations is a DHFR (-) cell line.

In one embodiment, one of the CHO cell populations of the plurality of CHO cell populations is a CHO GS cell line.

In one embodiment, a set of values is acquired for a plurality, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, of populations of CHO cells. In one embodiment, a set of values is acquired for a plurality of CHO cell populations that include a CHO Kl cell line, a CHO S cell line, a DG44 cell line and a DHFR (-) cell line.

As described elsewhere herein, an observation may be expressed in terms of the level of gene expression, for example, a gene described herein, or a metabolite described herein.

In one embodiment, the observations for each of the CHO cell populations include at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more observations of gene expression levels. In one embodiment, the observations for each of the cell populations include at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more observations relative to the levels of a glycan metabolite.

In one modality, one observation is the level of 4.4.1.0.0.

In one modality, one observation is the level of 4,4,1,1,0.

In one modality, one observation is the level of 4.5.1, 0.0.

In one modality, an observation is the level of 4,5,1,1,0.

In one modality, one observation is the level of 4,5,1,2,0.

In one modality, one observation is the level of 5.5.1.0.0.

In one modality, one observation is the level of 5,6,1,0,0.

In one modality, one observation is the level of 5,6,1,1,0.

In one modality, an observation is the level of 5,6,1,2,0.

In one modality, one observation is the level of 5,6,1,3,0.

In one modality, one observation is the level of 6,6,1,1,0.

In one modality, one observation is the level of 6,6,1,2,0.

In one modality, one observation is the level of 6,7,1,1,0.

In one modality, an observation is the level of 6,7,1,2,0.

In one modality, one observation is the level of 6,7,1,3,0.

In one modality, one observation is the level of 6,7,1,4,0.

In one modality, an observation is the level of a metabolite.

In one mode, one observation is the GDP-Fuc level.

In one aspect, the invention features a method of modeling a glycoprotein having a glycan complement and / or selected glycan component, or providing or selecting a population of CHO cells, for example, for use in the glycoprotein mode having a glycan complement and / or glycan component selected. The method includes: (i) acquire a profile of the cell population quality attribute (a profile), which comprises a set of responses, where a response is expressed in terms of a post-translational modification and is the product of an operation by a plurality of observations, for each of a plurality of populations of CHO cells, the acquired profiles forming a plurality of distinct profiles; (ii) acquiring the identity of the glycan complement and / or glycan component; (üi) compare the acquired profile with the identity acquired in (ii); (iv) in response to the comparison (eg, when the acquired profile and the acquired identity of the selected glycan component and / or glycan complement show a preselected relationship with each other, eg, the former includes the latter), select a of the plurality of CHO cell populations for production of the subject glycoprotein; selecting or providing the CHO cell population to perform the glycoprotein and / or performing the glycoprotein having the glycan component and / or glycan complement selected in the selected CHO cell population. In some embodiments, the method further comprises introducing a nucleic acid encoding all or part of the glycoprotein in the population of CHO cells.

In one embodiment, the method is a method for providing or selecting a population of CHO cells, for example, for use in the mode of a glycoprotein having a glycan complement and / or glycan component selected and the method further comprises culturing the CHO cell population.

In one embodiment, the method is a method of modeling a glycoprotein having a glycan complement and / or selected glycan component, and the method further comprises making a glycoprotein having a glycan complement and / or glycan component selected in the CHO cell population selected.

In one embodiment, the method may further comprise genetically modifying the selected CHO cell population to express the glycoprotein, for example, by introducing a nucleic acid encoding all or part of the glycoprotein in the identified CHO cell population.

In one embodiment, one of the CHO cell populations of the plurality of CHO cell populations is a DG4 cell line.

In one embodiment, a set of responses is acquired for a plurality, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, of populations of CHO cells. In one embodiment, a set of responses is acquired for a plurality of CHO cell populations that include a CHO Kl cell line, a CHO S cell line, a DG44 cell line and a DHFR (-) cell line.

In one embodiment, the observations for each of the CHO cell populations include at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more observations of gene expression levels. In one embodiment, the observations for each of the CHO cell populations include at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more observations relative to the levels of a glycan metabolite.

In one modality, one observation is the level of 4.4.1.0.0.

In one modality, one observation is the level of 4,4,1,1,0.

In one modality, one observation is the level of 4,5,1,0,0.

In one modality, an observation is the level of 4,5,1,1,0.

In one modality, one observation is the level of 4,5,1,2,0.

In one modality, an observation is the level of 5, 5, 1, 0, 0.

In one modality, one observation is the level of 5,6,1,0,0.

In one modality, one observation is the level of 5,6,1,1,0.

In one modality, one observation is the level of 5,6,1,2,0.

In one modality, one observation is the level of 5,6,1,3,0.

In one modality, one observation is the level of 6,6,1,1,0.

In one modality, one observation is the level of 6,6,1,2,0.

In one modality, one observation is the level of 6,7,1,1,0.

In one modality, an observation is the level of 6,7,1,2,0.

In one modality, one observation is the level of 6,7,1,3,0.

In one modality, one observation is the level of 6,7,1,4,0.

In one embodiment, an observation is the level of expression of a gene involved in a glycan biosynthesis.

In one modality, an observation is the level of a metabolite.

In one mode, one observation is the GDP-Fuc level.

As described elsewhere herein, various types of observations are suitable for use in the methods, for example, operations described herein, for example, an arithmetic combination or linear model.

As described elsewhere herein, various types of responses are suitable for use in the methods, for example, a response described herein, for example, a response that is the product of an operation at the expression level of a plurality of genes.

The titles and literals and numerals, for example, (a), (b), (i), etc., are presented merely to facilitate the reading of the description and claims. The use of titles and literals and numerals in the description or claims does not require that the steps or elements are carried out in alphabetical or numerical order or in the order in which they are presented.

Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

The figures are briefly described first: BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is an illustrative glycogram chromatogram of the isolated glycoprotein that was released, labeled and analyzed by LC and LC / MS; Fig. 2 is a representation of LC data illustrating the product distribution of CHO clones; Fig. 3 is a graph of PCA analysis for cell population quality attribute profiles (PACPC) for each of the cell types CHO Kl, CHO S, CHO DG44 and DHfr (-).

Fig. 4 is a representation of expression levels; Fig. 5 is a representation of expression levels; Fig. 6 is a linear model that uses the expression of ST3GAL3 to compute the glycan level 5,6,1,2,0 produced; Figs. 7A-7B are a representation of the distribution of transcripts related to glycosylation in the clones (each point) of each cell line background grouped for each transcript; Figs. 8A-8D are a representation of the analysis of PCA of transcripts of glycerelated genes derived from each of the clones from the bottoms of CHO cell lines, circles CH0K1, triangles CHOS, plus DG44; Figs. 9A-9D are a representation of the unknowns superimposed on the profiles of the quality attribute of the cell population for each of the four cell types.

DETAILED DESCRIPTION OF THE INVENTION Definitions As used herein, "acquiring a value" refers to any process that results in possession of the value. In one embodiment, the value is "acquired directly" by the one or more physical transformation steps, for example, in a sample, for example, a glycoprotein sample, a cell extract, or a cell sample, for example. , a cell line. In this way, the process results in a physical change in the sample or other substance, for example, an analytical reagent or a device used in the process. The methods, by way of example, include: analytical methods; preparatory methods; and cell manipulation, e.g., extraction or purification of components, e.g., nucleic acid, e.g., mRNA or DNA, or protein, from a cell, or from cultured cells. These methods typically include one or more of the following: separating a substance, eg, an analyte, or a fragment or other derivative thereof, from another substance; combining a substance, for example, an analyte, or a fragment or other derivative thereof, with another substance, for example a buffer solution, a solvent or a reagent; or changing the structure of an analyte, or a fragment of another derivative thereof, for example, by breaking or forming a covalent or non-covalent chemical bond, between a first and second atom of the substance, eg, an analyte . The value can also be "acquired indirectly". Indirect acquisition involves receiving the value, for example, from another party, for example a party that acquired the value directly. Typically, even in modalities characterized by indirect acquisition, either party has subjected a sample to a process such as those previously described, resulting in a physical change in the sample or other substance. In a modality, a part that practices the evaluation method instructs another party to perform the process, and for example, a part that practices the method receives the value. In one embodiment, a value can be the expression of whether a cell or a cell line has a characteristic or not or to what extent, for example, a characteristic related to the structure of a glycan, for example the level of a transcript, the capacity of creating a glycoprotein having a preselected glycan structure, a preselected level of a glycan structure, a preselected ratio of a first to a second glycan structure, or a preselected glycan structure in a preselected location.

A "cell population quality attribute profile" (PACPC) comprises a set of responses for a cell population. A set comprises at least two responses. Typically, a set comprises a response for a first cell, for example, a first isolate or aliquot of a cell population, and a response for a second cell, for example, a second isolate or aliquot of the cell population. A response, which is expressed in terms of a post-translational modification, for example, a glycan structure, is the product of the operation in a plurality of observations (for example, specific measurements or characteristics). An operation relates observations with a post-translational modification, for example, a glycan structure. In one embodiment, the observations are expressed in terms of a post-translational modification, for example, a glycan structure. In one embodiment the observation is not expressed in terms of a post-translational modification, for example, a glycan structure, for example, are expressed in terms of a gene expression, and the operation also converts them into units of a post-modification. ranslational, for example, a glycan structure. Examples of operations include a correlation of one or more observations with a post-translational modification, for example a glycan structure, for example by the use of a look-up table or an equivalent tool; the use of observations as inputs into the model, for example, a linear model, which relates observations with post-translational modifications, for example, a glycan structure; or, for example, when the observations are themselves expressed in terms of a post-translational modification, for example, a glycan structure, combination, for example, by addition, of observations. The observation can be obtained by analyzing the main components. The set of responses that comprise a profile of the quality attribute of the cell population, if considered as continuous, can be visualized / analyzed as if it defined a discrete space occupied by the cell population. For example, the set of responses can be described in n dimensions and occupy a space of n dimensions, for example, if they are described in 3 dimensions, the set defines a three-dimensional space.

In a "plurality of different profiles of the quality attribute of the cell population", as used herein, each profile of the quality attribute of the cell population in the plurality is different from each PACPC in the plurality, for example, at least one response from a first profile differs from at least one response from a second profile.

In one embodiment, a response is a direct indication of the state of a post-translational modification, for example, a glycostructure, for example, the presence or level of a glycostructure, a cell with an x level of glycan x and a level and glycan and . A selected post-translational modification, for example, a glycostructure, for example, a glycostructure present in a reference protein, is a post-translational modification, for example, a glycostructure, which will be included in a protein. If the set of responses includes the selected post-translational modification, for example, a glycostructure, or to put it another way, if the selected glycostructure falls within the profile of the quality attribute of the cell population, then the cell population can be selected to the production of a glycoprotein having the post-translational modification selected, for example, a glycostructure. The comparison of the post-translational modification, for example, a glycostructure, with a profile of the quality attribute of the plurality of the cell population allows for the selection of a cell population to optimize the production of a protein that has a post-modification. selected translational, for example, a glycostructure.

A "distinct isolate" as used herein, refers to a relationship between a first cell or group of cells and a second cell or group of cells. Different isolates have a common cell predecessor but where the founder cells of each distinct isolate are separated by at least 1, 10, 20, 50, 100, 500, 1,000, 5,000 or 10,000 cycles of cell divisions. To illustrate, a parental cell divides to form two Fl cells, each Fl cell divides to form two F2 cells, each F2 cell divides to form two F3 cells. There are three cycles of cell division between the parent cell and the F3 cell. Typically, the common cell predecessor is a cell, e.g., a cultured cell, that has been removed from a multicellular organism, e.g., an insect or an animal, e.g., a mammal or a primate, excluding as common cellular predecessors. the animal precursor cells or predecessors of the animal from which the common cell predecessor was taken.

An "observation", as used herein, is a value for the parameter, for example, a measure, a determined or observed value for a parameter, related to a property of a cell.

"Closely related cell populations", as used herein, refers to cell populations having one or more, and in two or more modes, or the totality, of the following properties: they are of the same species; they are of the same type of tissue; they are of the same cell type, for example, they are stromal cells; they have the same transformation state (for example, both are transformed and show essentially immortal growth in culture or both are incapable of immortalized growth, or both have growth rates that are within 2X of each other in a selected medium). In modalities, its founding cells separated from each other by less than 1,000 and, in modalities, by less than 500 or 100 cycles of cell division.

A "glycostructure," as used herein, refers to one or more elements of the glycan complement of a glycoprotein or to a selected glycan structure. It can refer, for example, to a single monosaccharide, to a single glycan component (for example, the presence of high-content structures in mannose), or to the glycan whole complement of a glycoprotein, or to a particular glycan structure, for example, to a high-duty glycan component.

The "glycan complement", as used herein, refers to all glycan components of a glycoprotein. In the case of a protein having a single glycosylation site, the glycan component bound thereto forms the glycan complement. In the case of a protein that has more than one glycosylation site, the glycan complement is created from glycan components bound to all sites. A "glycan complement component" refers to a subset of glycan components that form the glycan complement, for example, one or more glycan components bound to their respective glycosylation site or sites. The glycan complement may consist of the percentage of all the glycan components of all the glycoproteins in the mixture. The glycan component may also consist of all of the glycan components associated with a glycoform in a glycoprotein mixture.

The "glycan component" as used herein, refers to a sugar moiety, for example, a monosaccharide, oligosaccharide or polysaccharide (eg, a disaccharide, a trisaccharide, a tetrasaccharide, etc.) attached to a protein in a site. In embodiments, the linkage is covalent and the glycan component is linked by N or O to the protein. The glycan components can be monosaccharide chains linked together by means of glycosidic linkages. The glycan components can be linear or branched.

A "glycan structure", as used herein, refers to the structure of a glycan supplement, a component of a glycan supplement or a glycan component. The elements of a glycan structure include one or more of the following: the presence, absence or level of glycosylation at one or more sites, eg, one or more sites by N-linked or O-linked glycosylation; link by N u 0; length (number of monosaccharide residues); placement or location of a monosaccharide, for example, a galactosyl moiety, within a chain; saccharide content (for example, the amounts or proportions of monosaccharide components in a particular glycan); sequence of saccharides (for example, the order of subunits of monosaccharides in a glycan moiety); the presence, absence or quantity of a terminal or penultimate saccharide subunit; the number, placement and type (for example, the presence, absence or quantity of GlcNAc structures or bisect mañosa) of branch points; the presence, absence or level of a complex structure, for example, a biantenary structure, a three-year structure, a tetrantenary structure, etc .; the presence, absence or level of a high mañosa or a hybrid structure; the relationship between monosaccharide residues (e.g., linkages between monosaccharide residues, isomers and branching sites); the presence, absence, position or number of a selected monosaccharide, for example, a galactosyl moiety, a fucosyl moiety, a GlcNAc moiety or a mannosyl moiety; the presence, absence, position or number of a selected structure, for example, a mono-galactosylated, digalactosylated or polygalactosylated structure. Other non-exhaustive examples include any other structure found in natural glycoproteins; and heterogeneity or homogeneity at one or more sites (e.g., diversity throughout the protein, e.g., the degree of occupancy of potential glycosylation sites of a protein (e.g., the degree of occupancy of the same glycosylation site potential between two or more of the central structures of the particular protein in a plurality of molecules and the degree of occupancy of a potential glycosylation site in a central structure of the protein relative to different potential glycosylation sites in the same core structure of the protein) .

A glycan structure can be described in terms of a comparison of the presence, absence or amount of a first glycan structure with a second glycan structure. For example, the presence, absence or quantity of sialic acid in relation to the presence, absence or amount of fucose. In other examples, the presence, absence or amount of sialic acid such as N-acetylneuraminic acid can be compared, for example with the presence, absence or amount of a sialic acid derivative such as an N-glycolylneuraminic acid.

The glycan structures can be described, identified or tested in various ways. A glycan structure can be described, for example, in structural terms defined, for example, by the chemical name, or by a functional or physical property, for example, by molecular weight or by a parameter related to purification or separation, for example. , the retention time of a peak in a column or other separation device. In embodiments a glycan structure can be, by way of example, a peak or other fraction (representing one or more species) of glycan structures derived from a glycoprotein, for example from an enzymatic digestion.

A "monosaccharide" as used herein refers to the basic unit of a glycan component and in embodiments, a moiety that is transferred by a glycosyltransferase to a substrate. A "monosaccharide", as used herein, includes natural and non-natural monosaccharides. Examples of monosaccharide residues include glucose (Glc), N-acetylglucosamine (GlcNAc), mannose (Man), N-acetylmannosamine (ManNAc), galactose (Gal), N-acetylgalactosamine (GalNAc), fucose (Fue), sialic acid (NeuAc) ) and ribose, as well as derivatives and analogs thereof. Derivatives of different monosaccharides are known. For example, sialic acid encompasses over thirty derivatives with N-acetylneuraminic acid and N-glycolylneuraminic acid that form the core structures. Examples of sialic acid analogs include those that functionally mimic sialic acid, but are not recognized by siallases from endogenous host cells. Other examples of monosaccharide analogs include, but are not limited to, N-levulinoylmannosamine (ManLev), Neu5Aco-methylglycoside, NeuSAcp-methylglycoside, Neu5Aca-benzylglycoside, NeuSAc-benzylglycoside, Neu5Aca-methylglycoside methyl ester, Neu5Ac-methyl ester, acid 9-0-Acetyl-N-acetylneuraminic acid, 9-O-Lactyl-N-acetylneuraminic acid, N-azidoacetyl mannamine and 0-acetylated variations thereof and Neu5Aca-ethyl thioglucoside.

"High mannose" as used herein refers to one or multiple N-glycan structures that include HM3, HM4, HM5, HM6, HM7, HM8 and HM9 that contain 3, 4, 5, 6, 7, 8 or 9 trawl residues respectively.

Cells and cell lines The methods described herein utilize cells to produce glycoproteins having selected post-translational modifications (eg, glycostructures). Examples of cells and cell lines useful in these and other methods described herein follow below.

The cell useful in the methods described herein may be eukaryotic or prokaryotic, provided that the cell provides or has added thereto the appropriate enzymes to activate and attach (or remove) saccharides present in the cell or saccharides present in the medium. cell culture or fed to cells. Examples of eukaryotic cells include yeast, insect, fungus, plant and animal cells, especially mammalian cells. Suitable mammalian cells include any normal or abnormal normal or abnormal mortal animal or human cell, including: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293) (Graham et al., J. Gen. Virol. 36:59 (1977)); kidney cells of a baby hamster (BHK, ATCC CCL 10); Chinese Hamster Ovary (CHO), for example, DG44, DUKX-V11, GS-CHO (ATCC CCL 61, CRL 9096, CRL 1793 and CRL 9618); Mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HeLa, ATCC CCL 2); buffalo rat liver cells (BRL 3A; ATCC CRL 1442); human lung cells (138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse melanoma cells (NSO); mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather, et al., Annals N.Y. Acad. Sci. 383: 44 46 (1982)); canine kidney cells (MDCK) (ATCC CCL 34 and CRL 6253), HEK 293 (ATCC CRL 1573), WI-38 cells (ATCC CCL 75) (ATCC: American Type Culture Collection, Rockville, Md.), MCF-cells 7, MDA-MB-438 cells, U87 cells, A127 cells, HL60 cells, A549 cells, SP10 cells, DOX cells, SHSY5Y cells, Jurkat cells, BCP-1 cells, GH3 cells, 9L cells, MC3T3 cells, C3H- cells 10T1 / 2, NIH-3T3 cells, C6 / 36 cells, human lymphoblastic cell lines (for example GEX) and PER-Ce * cells. The use of mammalian tissue cell culture to express polypeptides is generally discussed in Winnacker, FROM GENES TO CLONES (VCH Publishers, N.Y, N.Y., 1987).

Examples of plant cells include, for example, Arabidopsis thaliana, rapeseed, corn, wheat, rice, tobacco, etc.) (Staub, et al., 2000 Nature Biotechnology 1 (3): 333-338 and McGarvey, P. B, et al., 1995 Bio-Technology 13 (13): 1484-1487; Bardor, M., Et al., 1999 Trends in Plant Science 4 (9): 376-380). Examples of insect cells include, for example, Spodoptera frugiperda Sf9, Sf21, Trichoplusia ni, etc. Examples of bacterial cells include Escherichia coli. Different yeasts and fungi such as Pichiapastoris, Pichia methanolica, Hansenula polymorpha, and Saccharomyces cerevisiae can also be selected.

Culture and processing media The methods described herein may include determining and / or selecting media components or culture conditions which result in the production of a desired glycostructure. The culture parameters that can be determined include media components, pH, feeding conditions, osmolarity, carbon dioxide levels, agitation rate, temperature, cell density, cell seeding density, elapsed time and spray rate.

Changes in production parameters such as the rate of agitation of a cell culture, the temperature at which the cells are grown, the components in the culture medium, the times at which the cultures start and end, variations in the time elapsed in the supply of nutrients can result in variations in the properties of a glycan of the produced glycoprotein product. Thus, the methods described herein may include one or more of: increasing or decreasing the rate at which the cells are agitated, increasing or decreasing the temperature at which the cells are grown, adding or removing components of means and alteration of times to which crops start or end.

Sequentially selecting production parameters or a combination thereof, as used herein, means that a first parameter (or a combination) is selected and then a second parameter (or combination) is selected, for example, based on a restriction imposed by the selection of the first production parameter.

Media The methods described herein may include determining and / or selecting a media component and / or the concentration of a media component that has a positive correlation with a desired glycostructure. A media component can be added or administered in the course of glycoprotein production or when a change occurs in the medium, which depends on the culture conditions. The media components include components added directly to the cultures, as well as components that are products derived from cell culture.

The media components include, for example, a buffer solution, amino acid content, vitamin content, salt content, mineral content, serum content, carbon source content, lipid content, nucleic acid content, content of hormones, trace element content, ammonia content, co-factors content, indicator content, small molecule content, hydrolyzate content and enzyme modulator content.

Physico-chemical parameters The methods described herein may include selecting culture conditions that correlate with a desired glycostructure. Such conditions may include temperature, pH, osmolarity, cutting force or agitation rate, oxidation, rate of euphorbias, growth vessels, tangential flow, DO, C02, nitrogen, fed batch, oxidative reduction, cell density and feeding strategy. Table 2 provides examples of physicochemical parameters that can be selected.

Table 2: DO temperature pH C02 Osmolarity Nitrogen Cutting force or rate of agitation Batch fed Oxidation Reduction oxidative Euphorbia rate Cell density Growing beakers Growing perfusion Tangential flow Strategy feeding Lot For example, the production parameter may consist of culturing a cell under conditions of acidic, neutral or basic pH. Temperatures can be selected from 10 to 42 ° C. For example, a temperature of about 28 to 36 ° C does not significantly alter the production of galactosylation, fucosylation, high-mannine production, hybrid production or sialylation of glycoproteins produced by a cell (e.g., a CHO cell, e.g. a CHO cell deficient in dhfr) grown at these temperatures. In addition, any method that slows down the growth rate of a cell can also have that effect. In this way, temperatures in this range or methods that reduce the growth rate can be selected when it is not desired to have these production parameters that alter the glycosynthesis.

In other embodiments, carbon dioxide levels can be selected, which results in a desired glycan characteristic or characteristics. The levels of C02 can be, for example, approximately 5%, 6%, 7%, 8%, 9%, 10%, 11%, 13%, 15%, 17%, 20%, 23% and 25% (and intermediate intervals). In one embodiment, when a decrease in fucosylation is desired, the cell can be cultured at C02 levels of about 11 to 25%, for example, about 15%. The C02 levels can be adjusted manually or can be derived from cells.

A wide variety of flasks, bottles, reactors and controllers allow the production and increase of cell culture systems. The system can be chosen based, at least in part, on its correlations with a desired property or glycan properties.

The cells can be cultured, for example, as a batch, fed batch, perfusion or continuous cultures.

The production parameters that can be selected include, for example, adding or removing the medium including when (at the beginning, middle or late during the culture time) and how often the medium is harvested; increase or decrease the speed at which cell cultures are agitated; increase or decrease the temperature at which the cells are grown; increase or decrease the medium in order to adjust the culture density; select a time in which cell cultures begin or end; and select a time in which the cell culture parameters are changed. The parameters can be selected for any batch, fed batch, perfusion and continuous culture conditions.

Glycoproteins The subject glycoproteins include natural and non-natural glycoproteins. Representative glycoproteins include: antibodies, eg, IgG, IgM, human, humanized, grafted and chimeric antibodies, and fragments thereof; fusion proteins, for example, fusions that include human (or other) antibody domains, e.g., Fe or constant region domains; growth factors; hormones, interferons; cytokines; cytokine receptors; soluble blood components, for example, albumin, coagulation factors, hematopoietic factors; enzymes; and any class of protein represented by a protein listed in Table 3. Soluble or active fragments of any of the glycoproteins or classes of glycoproteins described herein are also included.

Examples of glycoproteins that can be obtained by the methods described herein include those in Table 3 below: Table 3 In some embodiments, the methods described herein can be used to create glycoproteins with a selected level of high mannose, eg, a higher level of high mannose, compared to a reference glycoprotein.

EXAMPLES Example 1. Analysis of CTLA4 glycans produced in several isolates and the use of glycan data to distinguish cells with different backgrounds.

Four backgrounds of CHO cell lines were transfected with the gene encoding CTLA4IgG. These groups of cells were then subjected to selection and clonal selection to generate 20 clones from each of the four cell line backgrounds. The CTLA4IgG produced from each clone was isolated and purified by affinity chromatography of protein A. The glycans of the isolated glycoprotein were then released, labeled and analyzed by LC and LC / MS. In Figure 1 a chromatogram is illustrated. The illustrative LC data of the product distribution of each of the clones is described in Figure 2.

An analysis of the glycan data was used to distinguish the cells from the four backgrounds. The data on various aspects of the glycan structure were determined. Table 4 shows representative aspects of the glycan structure that can be used in this approach. In this table the glycans are represented as the composition of HexNAc, Hex, Fue, NeuAc, NeuGc, the presence of A, or B indicates the isomeric species and the presence of Ac indicates an acetylation event.

Table 4 The glycan data was then subjected to a Analysis of the Principal Components (ACP). The ACP provided the graphic representation shown in Figure 3. Although it is more easily represented as a rotary three-dimensional image, the angle 86 was chosen as a representative image of the ACP because it better illustrates the distribution of the clones in 2 dimensions. Surprisingly, this analysis provides a solid differentiation between the members of this group of relatively similar cell types. Surprisingly, the profile of the quality attribute of the cell population for each of the cell types, CHO Kl, CHO S, CHO DG44 and DHfr (-) are not only different, but they allow an unambiguous selection of a cell line that has a desired quality, for example, as shown by the differentiation along the X axis.

Example 2. Correlation of glycan structures with gene expression data using linear models.

Four lines of Chinese hamster ovary cells were transfected with a gene to produce the CTLA4-Ig protein. The clones of each cell line were obtained by cloning; Clonal cell lines were expanded so as to produce the CTLA4-Ig protein for glycan and RNA analysis for analysis of gene expression. Cellular CTLA4-Ig RNA and protein were obtained from 20-24 clones of each cell line. The messenger RNA (mRNA) was analyzed by RT-PCR to measure the expression levels of 28 genes related to glycosylation. The expression levels of genes related to glycosylation were normalized by one or more domestic genes (eg, β-actin or ribosomal protein genes). Levels of linearized expression were obtained by an exponential transformation of the normalized expression level of the housekeeping gene. These data are illustrated in Figure 4. The glycans were obtained from the CTLA4-Ig protein and analyzed by several methods, including LCMS / MS. A percentage composition was calculated for each glycan species. The representative data are shown in Figure 5.

Linear modeling was used to discover relationships between glycan structures and gene expressions. The discovery of the linear model was performed with the computer environment R using the following method. For each measured glycan, the data set was divided into training and test sets using a resampling analysis with a stratification method to ensure equal representation of isolates from the four cell lines. The best suitable coefficients of the linear model for each individual gene were computerized and recorded for the training set; the model adjustment error was recorded. Gene expression levels were used to calculate the glycan level for samples in the test set the estimation error was recorded. The linear model with the best fit for the training set was retained. All models of two genes were evaluated by the addition of each remaining gene to the best fit model of a gene. The model of two genes of better fit was retained. This process was repeated until models of 10-15 genes were generated. The complete process was repeated from the generation of training and test sets by 20 iterations for each glycan in order to measure the repeatability of the discovery of the best fit models.

Subsequently, a detailed analysis of the model was carried out. Models that use more than 5 genes were determined undesirable due to the universally high error rates for test sets that indicate overadjusted data. For each glycan, the frequency of occurrence was computed from a particular gene in the first five positions of the 20 series of model discoveries. The genes that appear most frequently were selected for a detailed analysis of the model in which 200 training iterations and error rates of test sets were computed using resampling analysis with stratification with subsequent calculation of the coefficient for the best fit linear model using the target genes. Errors were recorded for training and test sets for each iteration. Models with desirable training and test errors were subsequently compared with each other by fitting the model to the full data set that performs F tests of model errors to justify the selection of more complex models rather than simple models.

In the example included here (see Figure 6), a linear model that uses an ST3GAL3 expression to compute glycan level G5.6.1.2.0 produced a reasonable fit at the glycan measurement level. A linear model with ST3GAL4 did not produce a model with an appropriate fit. However, the addition of ST3GAL4 to the ST3GAL3 model produced a model with a considerably better fit model for the data according to F tests (p = 0.0011). The sign of the coefficients for the two genes indicates that a higher expression of ST3GAL3 increases the level of G5.6.1.2.0 and a higher expression of ST3GAL4 decreases the level of G5.6.1.2.0. This relationship was unexpected.

Variance Analysis Table Model 1: G5.6.1.2.0 ~ ST3GAL3 Model 2: G5.6.1.2.0 - ST3GAL3 + ST3GAL4 Grad. Lib. beef. Grad. Lib. SRC Sum of quad. F Pr (> F) 1 29 30.5688 10 2 28 20.7739 1 9.7949 13.202 0.001112 ** fifteen 9 Example 3. Variability and classification of cell lines.

Four Chinese hamster ovarian cell lines were transfected with a gene to produce CTLA4-Ig protein. The clones of each cell line were obtained by cloning by dilution; Clonal cell lines were expanded so as to produce the CTLA4-Ig protein for glycan and RNA analysis for gene expression analysis. Cellular CTLA4-Ig RNA and protein were obtained from 20-24 clones of each cell line. The messenger RNA (mRNA) was analyzed by RT-PCR to measure the expression levels of 28 genes related to glycosylation. The expression levels of genes related to glycosylation were normalized by one or more housekeeping genes (ie, β-actin or ribosomal protein genes). Linearized expression levels were obtained by an exponential transformation of the normalized expression level of the housekeeping gene.

The profiles of transcriptional data for a variety of genes related to glycosylation are illustrated in Figure 7. Figure 7 represents the distribution of transcripts related to glycosylation in all clones (each point) of each cell line antecedent (Blue, Red, Green or Black) grouped for each transcript. The genes followed were the following: the transcripts A1-A8, Bl-5, C5, 6 are glycosyltransferases; B6-8, Cl-4, Dl-4, are from biosynthetic enzymes; C7,8, D5,6, are normalization transcripts and CTLA4IgG. The transcriptional data were then subjected to a Principal Component Analysis (PCA) blind to the identifications of cell line backgrounds. The first three main components were plotted on the x, y and z axes. The clones were then assigned a symbol according to their cell line origin as illustrated in Figure 8. Surprisingly, the profile of the quality attribute of the cell population for each of the cell types, CHO Kl, CHO S, CHO DG44 and DHfr (-) are not only different, but allow unambiguous selection of a cell line having a desired quality, for example, as shown in Figure 8.

Then a blind assay was carried out in which the transcriptional profile was measured for 21 cell isolates of unknown origin. The origin of each cell line was unknown to researchers. However, they knew that they possessed the potential to be derived from any CHO cell line, Kl, S, DG44 and DHfr (-). The data of the isolates of unknown origin were transformed into the coordinate system used in the ACP of the original data and plotted together with the original data. See Figure 9, which shows the overlapping strangers in the profiles of the quality attribute of the cell population for each of the four cell types derived from known origins. The identity of each cell line was prela by linear discriminatory analysis (APL); 20 of 21 clones were correctly classified. The profiles of the quality attribute of the cell population allowed a correct assignment of one of the four cell types to 20 of 21 isolates of unknown cells.

Extensions and alternatives All bibliography and similar material cited in this application, including, but not limited to, patents, patent applications, articles, books, treaties and web pages, regardless of the format of the bibliography or similar materials , are incorporated herein by reference in their entirety. In case one or more of the bibliographies or similar materials incorporated differ from this application or contradict it, including, but not limited to, the defined terms, the terms of use, the techniques described or similar, this request will prevail. The titles of sections used herein are included solely for organizational purposes and should not be construed as limiting the described purpose in any way. While the methods have been described along with different modalities and examples, the methods are not intended to be limited to modalities or examples. On the contrary, the methods encompass different alternatives, modifications and equivalents, as will be appreciated by those skilled in the art.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for carrying out a glycoprotein having a glycan complement or selected glycan component characterized in that it comprises: (a) acquire the identity of a cell population for the production of the glycoprotein, where the identity is acquired or determined by (i) acquisition, for each of a plurality of isolates or aliquots of a first cell population, of a value that is expressed in terms of a glycan complement or a glycan component, whose value is a function of a plurality of different observations that include the level of expression of a plurality of different genes and the level of expression of a plurality of different glycostructures, glycan structures, glycan components, or combinations thereof to provide a set of values for the first cell population; (ii) acquisition, for each of a plurality of isolates or aliquots of a second cell population, of a value that is expressed in terms of a glycan complement or a glycan component, whose value is a function of a plurality of different observations that include the level of expression of a plurality of different genes and the level of expression of a plurality of different glycostructures, glycan structures, glycan components, or combinations thereof to provide a set of values for the second cell population; (iii) comparison of a value for a complement of selected glycan or glycan component with a set of values of the first cell population and with the set of values for the second cell population; Y (iv) in response to the comparison, selection of the first or second cell population; Y (b) culturing the selected cell population to thereby perform the glycoprotein having the glycan complement or glycan component selected.

2. The method according to claim 1, characterized in that it further comprises isolating the glycoprotein from the culture.

3. The method according to claim 2, characterized in that it further comprises purifying the glycoprotein.

4. The method according to claim 1, characterized in that it also comprises: (i) acquire a profile of the quality attribute of the cell population (a profile) comprising a set of responses, where a response is expressed in terms of a glycan complement or a glycan component and is the product of an operation in a plurality of observations, for each plurality of cell populations, the acquired profiles form a plurality of different profiles; (ii) acquiring the identity of a glycan supplement or a selected glycan component; (iii) compare the acquired profile with the identity acquired in (ii); (iv) when the acquired profile includes the identity acquired in (ii), select one of the pluralities of cell population to perform the glycoprotein having the glycan complement or glycan component selected.

5. The method according to claim 1, characterized in that it further comprises introducing a nucleic acid that encodes all or part of the glycoprotein within the identified cell population.

6. A method for carrying out a glycoprotein having a glycan complement or selected glycan component, characterized in that it comprises: (a) acquire the identity of a population of CHO cells for the production of the glycoprotein, wherein the identity is acquired or determined by (i) acquisition, for each of a plurality of isolates or aliquots of a first population of CHO cells, of a value that is expressed in terms of a glycan complement or a glycan component, whose value is a function of a plurality of different observations that include the level of expression of a plurality of genes or metabolites and the level of expression of a plurality of different glycostructures, glycan structures, glycan components, or combinations thereof to provide a set of values for the first CHO cell population; (ii) acquisition, for each of a plurality of isolates or aliquots of a second population of CHO cells, of a value that is expressed in terms of a glycan component or a glycan complement, whose value is a function of a plurality of different observations that include the level of expression of a plurality of genes or metabolites and the level of expression of a plurality of different glycostructures, glycan structures, glycan components, glycan complement or combinations thereof to provide a set of values for the second population of CHO cells where the second population of CHO cells digests from the first population of CHO cells in a naturally acquired or intentionally induced mutation; (iii) comparing a value for a glycan component or selected glycan complement with the set of values of the first population of CHO cells and with the set of values for the second population of CHO cells; (iv) in response to the comparison, selection of the first or second population of CHO cells; Y (b) culturing the selected CHO cell population to thereby perform the glycoprotein having the glycan component or glycan complement selected.

7. The method according to claim 6, characterized in that the observation is one or more of the level of 4,4,1,0,0; the level of 4,4,1,1,0; the level of 4,5,1,0,0; the level of 4,5,1,1,0; the level of 4,5,1,2,0; the level of 5.5.1.0.0; the level of 5,6,1,0,0; the level of 5,6,1,1,0; the level of 5,6,1,2,0; the level of 5,6,1,3,0; the level of 6,6,1,1,0; the level of 6,6,1,2,0; the level of 6,7,1,1,0; the level of 6,7,1,2,0; the level of 6,7,1,3,0; the level of 6,7,1,4,0; the level of expression of a glycosyltransferase; the level of expression of a gene that participates in glycan biosynthesis; the level of metabolites; the level of U P; the level of GTP; the level of UDP-Gal; the level of GDP-Fuc.

8. The method according to claim 1, characterized in that a set of values is acquired by a plurality of CHO cell populations that include a CHO Kl cell line, a CHO S cell line, a DG44 cell line and a line of CHO cells. DHFR cells (-).

9. A method for providing or selecting a cell population of a plurality of isolates from a cell population for use in the form of a glycoprotein having a glycan complement or a selected glycan component, characterized in that it comprises: (a) acquiring the identity of a glycan supplement or a selected glycan component; (b) acquiring an assessment of the ability of each of the pluralities of isolates of the cell populations to produce the glycan complement or glycan component, and (c) selecting an isolate from the plurality of isolates, to thereby provide an isolate of a cell population for use in the form of a glycoprotein having a glycan complement or selected glycan component.

10. A method for monitoring a production process to perform a glycoprotein having a selected post-translational modification, characterized in that it comprises: (a) acquiring, for each of a plurality of isolates or aliquots of a first cell population, a value that is expressed in terms of a glycan complement or a glycan component, whose value is a function of a plurality of different observations which include the level of expression of a plurality of different genes and the level of expression of a plurality of different glycostructures, glycan complement or glycan component to provide a set of values for the first cell population; (b) identifying a glycan supplement or selected glycan component; (c) comparing a value for the complement of glycan or selected glycan component with the set of values of the first cell population; Y (d) if the comparison shows that the set of values for the first cell population includes the value for the complement of glycan or selected glycan component, look for a first option, for example, continue the culture; and if the comparison shows that the group of values does not include the value for the glycan supplement or glycan component selected, look for a second option, for example, cease the current culture conditions or grow under a new set of conditions.

11. A method for selecting a glycoprotein for manufacturing in a cell population, characterized in that it comprises: (a) acquire a profile of the quality attribute of the cell population, comprising a set of responses, wherein a response is expressed in terms of a glycan complement or a glycan component and is the product of an operation in a plurality of observations, for a cell population; (b) acquiring the identities of a plurality of glycan or glycan component complement; (c) compare the acquired profile with the identity acquired in (b); (d) when the identities acquired in (b) include the acquired profile, select one of the plurality of glycan complement or glycan component for production in the cell population; Y (e) carrying out a glycoprotein having the selected glycostructure in the cell population.

12. A database comprising a plurality of records for isolates from a cell population of a preselected cell population, characterized in that each record comprises an identifier for a single asylee of the preselected cell type and an identifier of a profile of the quality attribute of the cell population for the asylee, and where the profile of the quality attribute of the cell population for each entry is unique compared to others in the plurality for the isolate.